Rapid Enzyme-Linked Immunosorbent Assay for Detection of the Algal Toxin Domoic Acid

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Journal of Shellﬁsh Research, Vol. 27, No. 5, 1301–1310, 2008.

RAPID ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF THE ALGAL TOXIN DOMOIC ACID

R. WAYNE LITAKER,1* THOMAS N. STEWART,2 BICH-THUY L. EBERHART,3 JOHN C. WEKELL,3 VERA L. TRAINER,3 RAPHAEL M. KUDELA,4 PETER E. MILLER,4 ALICE ROBERTS,4 CASSANDRA HERTZ,4 TYLER A. JOHNSON,4 GREG FRANKFURTER,5 G. JASON SMITH,5 ASTRID SCHNETZER,6 JOE SCHUMACKER,7 JONNETTE L. BASTIAN,7 ANTHONY ODELL,8 PATRICK GENTIEN,9 DOMINIQUE LE GAL,10 D. RANSOM HARDISON1 AND PATRICIA A. TESTER1 1National Ocean Service, NOAA, 101 Pivers Island Road, Beaufort, North Carolina 28516; 2Mercury Science Inc., 2801 Blue Ridge Road, Suite G-70, Raleigh, North Carolina 27607; 3Northwest Fisheries Science Center, NOAA, 2725 Montlake Boulevard East, Seattle, Washington 98112; 4Ocean Sciences & Institute for Marine Sciences, University of California Santa Cruz, 1156 High Street, Santa Cruz, California 95064; 5Moss Landing Marine Laboratories, 8272 Moss Landing Rd, Moss Landing, California 95039; 6Department of Biological Sciences, University of Southern California, 3616 Trousdale Parkway, AHF 301, Los Angeles, California 90089; 7Quinault Indian Nation, Division of Natural Resources, Department of Fisheries, 1214 Aalis Drive Bldg C, Taholah, Washington. 98587; 8Olympic Region Harmful Algal Bloom Partnership, 14 North Shore Place, Hoquiam, Washington 98550; 9IFREMER- Centre de Brest, DYNECO, Pointe du Diable, BP 70, 29280 Plouzane, France; 10IFREMER, 13 Rue Kerose, 29900 Concarneau, France

ABSTRACT Domoic acid (DA) is a potent toxin produced by bloom-forming phytoplankton in the genus Pseudo-nitzschia, which is responsible for causing amnesic shellfish poisoning (ASP) in humans. ASP symptoms include vomiting, diarrhea, and in more severe cases confusion, loss of memory, disorientation, and even coma or death. This paper describes the development and validation of a rapid, sensitive, enzyme linked immunosorbent assay test kit for detecting DA using a monoclonal antibody. The assay gives equivalent results to those obtained using standard high performance liquid chromatography, fluorenylmethoxycarbonyl high performance liquid chromatography, or liquid chromatography—mass spectrometry methods. It has a linear range from 0.1–3 ppb and was used successfully to measure DA in razor clams, mussels, scallops, and phytoplankton. The assay requires approximately 1.5 h to complete and has a standard 96-well format where each strip of eight wells is removable and can be stored at 4C until needed. The first two wells of each strip serve as an internal control eliminating the need to run a standard curve. This allows as few as 3 or as many as 36 duplicate samples to be run at a time enabling real-time sample processing and limiting degradation of DA, which can occur during storage. There was minimal cross-reactivity in this assay with glutamine, glutamic acid, kainic acid, epi- or iso-DA. This accurate, rapid, cost-effective, assay offers environmental managers and public health officials an effective tool for monitoring DA concentrations in environment samples.

KEY WORDS: ASP, domoic acid poisoning, ELISA, mussels, scallops, razor clams, test kit

INTRODUCTION has been shown to commonly accumulate in the edible parts of razor clams (Siliqua patula), mussels (Mytilus californianus or Domoic acid (DA) is a potent toxin produced by bloom- edulis), and Dungeness crabs (Cancer magister) (Wekell et al. forming phytoplankton in the genus Pseudo-nitzschia (Fig. 1). It 1994, Horner et al. 1997). High levels of DA in razor clams in is a glutamate analog, which acts as a potent excitatory Oregon and Washington are responsible for beach closures that neurotransmitter and causes amnesic shellfish poisoning (ASP) can last for more than a year. Losses of more than $20 million in humans (Quilliam & Wright 1989, Quilliam et al. 1989b, annually result from these closures caused by lost tourism and Wright et al. 1989). Symptoms include vomiting, diarrhea, and in reduced recreational and commercial and tribal clam harvests more severe cases confusion, loss of memory, disorientation, and (Adams et al. 2000). DA has also been implicated in the death even death. As a tricarboxylic acid, fully ionized at seawater pH, and illness of brown pelicans (Pelecanus occidentalis)and DA can behave as a potent trace metal ligand (Rue & Bruland Brandt’s cormorants (Phalacrocorax penicillatus)(Fritzetal. 2001, Wells et al. 2005). DA can bioaccumulate and rapidly 1992, Work et al. 1993), California sea lions (Zalophus califor- transvectors throughout the food chain via clams, mussels, crabs, nianus) (Scholin et al. 2000, Trainer et al. 2000, Brodie et al. filter feeding fish, and other organisms (Horner & Postel 1993, 2006), sea otters (Enhydra lutris) (Kreuder et al. 2003), and Scallet et al. 2005, Vigilant & Silver 2007). DA poisoning was first possibly whales (Lefebvre et al. 2002). recognized after a lethal event on Prince Edward Island, Canada The regulatory method for DA detection sanctioned by the in 1987 (Wright et al. 1989). Since that time, a number of toxic Interstate Shellfish Sanitation Conference (Quilliam et al. events have occurred on the United States west coast where DA 1989a, Quilliam et al. 1995) is a high performance liquid chromatography (HPLC) assay (Quilliam et al. 1991, Hatfield *Corresponding author. E-mail: [email protected] et al. 1994). Though accurate, these analyses are generally run

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1302 LITAKER ET AL. by centralized state facilities with results typically not available are causes for public health concern. The average cost of for 3–14 days after the samples are collected. In more remote approximately $100 per sample limits the number of samples communities, many of which depend heavily on subsistence that can be analyzed (Harold Rourk, WA State Department of clam harvests, these long delays and the costs of sample analysis Health, pers. comm.). Resource managers in coastal communities have expressed their desire for a cost-effective method for rapid and accurate determination of DA concentrations in shellﬁsh and phytoplankton samples. This paper describes the development and optimization of a robust monoclonal antibody based enzyme-linked immunosorbent assay (ELISA) test kit for DA that will meet management needs for rapid detection of DA in environmental samples.

MATERIALS AND METHODS

Assay Kit Overview

The DA assay kit was developed jointly by NOAA’s National Centers for Coastal Ocean Science, National Ocean Service, and the Northwest Fisheries Science Center, together with an industry partner Mercury Science, Inc., Durham, NC (NOAA/MSI). It was designed as a sequential competitive enzyme linked immunosorbent assay (ELISA) utilizing a high avidity monoclonal antibody (mAb) to DA to ensure assay specificity and consistency across production lots. In the current format, a fixed number of anti-DA mAb binding sites are incubated with dissolved DA in the sample followed by the addition of a DA—horseradish peroxidase (HRP) conjugate. As these binding events occur, the anti-DA mAb molecules are simultaneously captured by antimouse antibodies affixed to the surface of the microtiter plate wells. Subsequent HRP derived color development, readable on standard microplate readers, was inversely proportional to the concentration of DA in the sample matrix. The assay reagents were titrated so that the amount of mAb and the DA–HRP conjugate added produced a maximal absorbance signal of 3 absorbance units when no DA was present. The implementation of this ELISA system required the development and validation of two essential reagents, a high avidity monoclonal antibody to DA and a stable DA-HRP conjugate recognized by the same mAb.

Production of the Anti-Domoic Acid Antibody

Domoic acid (Sigma-Aldrich, St. Louis, MO), was conju- gated with bovine serum albumin (BSA) using dicyclohexyl carbodiimide and N-hydroxysuccinimide by a two-step syn- thetic pathway (Adamczyk et al. 1994). Ten mice were immu- nized with the DA-BSA immunogen. Serum titers were determined five days after each boost. A fusion was performed on the three mice that showed the greatest response. Hybridoma cell lines and monoclonal antibody production was performed according to the method of Fenderson et al. (1984). The 10 clones with highest affinity mAbs were selected for further growth and their affinity to DA was compared. The most sensitive clone was ultimately selected as the primary mAb for use in the assay development.

DA-HRP Conjugate

Domoic acid (Sigma) was cross-linked to horseradish peroxidase (HRP) using the procedure of Yoon et al. (1993). The Figure 1. Structure of domoic acid, the isomers epi-domoic acid, iso- reagent was tested for stability and was used to screen for high domoic acid, and two analogues kainic acid and glutamic acid. afﬁnity mAbs after the fusion and for assay development. JOBNAME: jsr 27#5 2008 PAGE: 3 OUTPUT: Friday November 7 23:26:15 2008 tsp/jsr/176293/27-5-06

DOMOIC ACID TEST KIT 1303

Domoic Acid Standards the concentration ratio of anti-DA antibody and DA–HRP conjugates are standardized within reagent lots, the kinetics of The DA standards used to calibrate the assay were pur- the reaction were ﬁxed between assay runs (assuming constant chased from the Certiﬁed Reference Materials Program at the temperature), such that the slope and ED values remain National Research Council of Canada Institute for Marine 50 constant. This made it possible to calculate DA concentrations Biosciences (Halifax, Nova Scotia, Canada). using the four parameter model.

Assay Calibration slope DA concentration ¼ ED50½ðBo=BÞ1

A series of dose response curves using varying amounts of Because the slope and ED50 are constants, all that was needed to antibody and DA-HRP were performed to optimize the assay calculate the DA concentrations was an accurate Bo and the B sensitivity. The optimal assay conditions were found to have an estimates from individual samples. In the assay, the mean value effective linear range from approximately 0.1–3.0 ppb. These for Bo for each strip of wells was determined by adding sample conditions were used in all the subsequent phases of assay dilution buffer lacking DA to the first two wells in that strip. development. The antibody was also tested for cross-reactivity Duplicate aliquots from each of three extracted samples diluted with varying concentrations of kainic acid, glutamine and with sample buffer were then added to the six remaining wells to glutamic acid. These compounds are structurally similar to obtain the B values. Duplicates were run to ensure assay various portions of DA molecule and have the potential to replicability. It should be noted that Bo (the maximal value cross-react with anti-DA mAbs. Glutamine and glutamic acid, with no DA added) can have noticeable variation between in particular, are common in animal tissues, including shellfish. assays depending on differences in temperature and development time as shown in Figure 2A. However, when the B values Calculation of the Parameters Needed to Construct an Internal Domoic for each strip are divided by Bo, the kinetics of the curve become Acid Standard for Each Well Strip normalized (i.e., replicable between strips and between runs) (see Fig. 2B). In this way the average B values serves as an Using the optimized DA assay, multiple dose response o internal standard that can be used in place of a standard curve curves were made using the NRC standards diluted to between provided the variation in the B is not above or below certain 0 and 10 ppb (1–10 ng mL–1) in the assay reaction buffer. The o limits, which are specified in the calculation software described average response derived from each of the individual response later. curves was calculated and a dose response curve was generated using a four parameter logit-log curve fitting analysis (Ritchie Domoic Acid ELISA Test Kit Procedure et al. 1981; Fig. 2). Four parameters were derived from this analysis. This first was Bo, the maximal signal, which occurred The 96-well assay tray used in the assay contained 12 strips. when no sample DA was present (Fig. 3A). The second was B, Each strip of 8 wells could be removed and stored until it was the signal produced by a known amount of sample DA. The needed. The first two wells of each strip were used as a control third was the slope of the logistic transformed data [propor- (no DA added). The remaining six wells were used to analyze tional to the linear portion of the sigmoidal curve describing the three samples in duplicate. This format provided the flexibility relationship between the ln sample DA concentration versus of running anywhere from 3–36 duplicate samples at a time. For signal (B)]. And the fourth was ED50, the DA concentration at unknown sample analysis, extracts were diluted to a final the mid point of the slope curve where half the available anti- concentration ranging from 0.3–3 to ppb using the sample DA mAbs in the well are bound to DA-HRP (Fig. 3A). Because buffer (phosphate salt solution, pH 7.8, containing casein). For

Figure 2. (A) Representative dose response curves for domoic acid analyzed on different days. It should be noted that Bo (the average of the maximal 450 nm absorbance values from the first two wells of a strip to which no DA is added) can vary noticeably between assays depending on differences in ambient temperature and development time. (B) The mean and SD in signal from eight normalized domoic acid dose response curves carried out over the course of several weeks. These data were specifically normalized by dividing each of the resultant absorbance values by Bo. The result of this normalization process, given that the concentrations of antidomoic acid antibody and HRP-domoic acid conjugate are fixed, is that the resultant curves are replicable between rows and between assays done on different days. The black squares and error bars indicate the mean value at each given domoic acid concentration %1SD. JOBNAME: jsr 27#5 2008 PAGE: 4 OUTPUT: Friday November 7 23:26:17 2008 tsp/jsr/176293/27-5-06

1304 LITAKER ET AL.

temperature for 30 min on an orbital shaker set to vigorously mix the solution in each well (PerkinElmer Waltham, MA 1296–004 DELFIA Plateshake set on high). Vigorous mixing is key to obtaining replicable results from one run to the next. In this step, the bulk of the native DA will bind to available mAbs in proportion to the DA concentration. At the end of the incubation, 50 mL of DA HRP conjugate was added to each well and the plate incubated a second time for 30 min at room temperature on an orbital shaker. The DA-HRP will then bind to remaining available mAb sites. After the incubation, the plate was washed three times with wash solution [Tris-HCl buffered salt solution (pH 7.8) containing Tween 20 and sodium azide as a preservative] using a commercial plate washer, making certain the fluid was completely aspirated from all the wells. Alternatively, these washes can be done manually by adding wash solution to wells using a multichannel pipettor and then flicking all fluid from the wells. The manual method may result in slightly higher variability. Next, 100 mL of K-Blue TMB substrate (5.5#-tetramethylbenzidine, Neogen Corpora- tion, Lexington, KY) was added to each well. The plate was placed on an orbital shaker for no more than 5 min, or until adequate color development was observed. Color development was terminated by adding 100 mL stop solution (1N hydro- chloric acid) to each well. The absorbance in each well was measured at 450 nm using a Thermo Ascent MultiSkan plate reader (Thermo Scientific, Waltham, MA). The DA concentrations were determined using the sample (B) and control (Bo) absorbances, the original tissue weights, and the volume of 20% or 50% methanol used to extract each sample. The actual calculations were made using a Microsoft Excel work sheet (Microsoft Corporation, Redmond, WA), which incorporates the constants for the four parameter model described above. This worksheet can be downloaded from Stewart (2008). Processing time for this assay was ;1.5 h.

Routine Tissue Extraction

In the case of razor clams and scallops, pooled samples of Figure 3. (A) DA concentrations versus the corresponding ELISA 10–12 individual shellfish were cleaned, and ground to a smooth absorbance values, which were normalized by dividing by maximal (Bo) and uniform homogenate in a commercial blender (Waring absorbance value. (B) Log-logit transform of the data shown in Fig. 3A. model HGBSS56, Torrington, CT). Clams were pooled because From this analysis it was possible to calculate the parameters needed to previous studies of DA in razor clams from the Washington accurately calculate domoic acid concentrations using the ELISA assay. coast indicated that the coefficient of variation for DA between These parameters include Bo, the maximal absorbance value at 450 nm obtained from the first two wells of a strip to which no free domoic acid is clams in a population exceeded 100% (Wekell et al. 2002). If the added and B, the 450 nm absorbance value for a given sample, slope of the homogenate appeared to be forming a gel caused by unusually logit-log transformed data, which were proportional to the linear portion high lipid content, an equal weight of water was added and the of the sigmoidal curve describing the relationship between the ln DA dilution noted. Approximately 2 g of homogenized tissue were concentration versus signal (B), and ED50, the mid point of the slope curve added to a tared 50 mL conical tube and the weight recorded to where half the available anti-DA mAbs are bound to DA. the nearest 0.01 g. Next, 18 mL of 50% methanol were added and the samples mixed at high speed on a vortex mixer for 2 min. Once the extraction was completed the tubes were spun in clam tissues containing DA, sample dilutions of 1:50 and 1:1000 a table top centrifuge for 20 min at 10,000 3 g or until a tight were typically used. Preliminary tests with razor clam extracts pellet and clear supernatant were obtained. If the samples did showed that a 25-fold dilution in sample dilution buffer not clear despite the spinning at high speed, the supernatant was eliminated matrix effects in ELISA analysis. poured into a syringe, then passed through a 0.45 mm Millex HA The assay was initiated by adding 50 mL of the anti-DA syringe filter (Millipore, Billerica, MA) to remove proteins and antibody to each well using a multi channel pipettor. Next, 50 other compounds that can form micelles, whereas soluble DA mL of the control solution (sample buffer without DA) was remained in the filtrate. At this point the homogenate was ready added to the first two wells in each row. Duplicate 50 mL for analysis by ELISA and HPLC. If necessary, the sample was aliquots from the diluted DA extracts were then added to the stored at 4C for up to 24 h in an explosion proof refrigerator remaining wells in each strip and the plate incubated at room prior to analysis. JOBNAME: jsr 27#5 2008 PAGE: 5 OUTPUT: Friday November 7 23:26:20 2008 tsp/jsr/176293/27-5-06

DOMOIC ACID TEST KIT 1305

Phytoplankton Extraction column. The columns were washed with 5 mL of 0.1 M NaCl in 10% aqueous acetonitrile (10% acetonitrile: 90% deionized Approximately 0.1–1.0 L of cultured cells or sea water water). The columns were immediately moved to a new row in samples were filtered onto a GF/F filter, which was immediately the vacuum manifold and the DA eluted from the SPE cartridge frozen at –80 C until the filter could be processed. For process- using 5 mL of 0.5 M NaCl in aqueous 10% acetonitrile (10:90, ing, the filter was placed in a 5 mL conical BD Falcon Tube acetonitrile:deionized water) and collected in 5 mL graduated (Becton Dickinson, Franklin Lakes, NJ) and 3 mL of 20% centrifuge tubes. Flow was stopped when eluant reached 4.9 mL methanol were added. The samples were then sonicated using a in the graduated centrifuge tube. The graduated centrifuge tube Thermo Fisher Scientific Model 100 Sonic Dismembrator with was removed from the manifold and the actual volume a 1/8 inch probe (model 15-338-80, Fisher Scientific, Waltham, recorded. The graduated centrifuge tubes were capped and the MA) until the filter was completely homogenized. Care was eluant immediately mixed by shaking the tube vigorously 5–10 taken to prevent the probe from rupturing the tube. The times. Tissues from the other invertebrate species examined sonicator probe was cleaned very carefully with 20% methanol (Table 1) were processed similarly, except that the extracts were between samples to prevent cross-contamination. Next the filtered through Nanospec MF GHP 0.45 mm centrifugal filters homogenate was centrifuged at 3000 g for 10 min. The (Pall, Ann Arbor, MI) instead of SPE columns before HPLC supernatant was then passed through a disposable Whatman analysis. Eluted samples were transferred to HPLC analysis GD/X 0.2 m syringe filter (Florham Park, NJ) into a 5 mL m vials. The HPLC conditions were as follows: Vydac TP210 tube. At this point the sample was split for analysis using both column (Grace, Deerfield, IL), 2.1 by 250 mm, 40C, elution of the ELISA and HPLC assays. DA in 10% acetonitrile containing 0.1% trifluoroacetic acid (TFA). Twenty ml of each sample were injected into the column HPLC Validation of DA concentration from Razor Clam Tissues and eluted isocratically at 0.3 mL per min. The retention time for the DA peak was about 6–8 min depending on the column. HPLC is the accepted standard method for measuring DA Canadian NRC DACS standards at concentration of 1 ppm in and is the basis of the current official method for regulatory 10% acetonitrile solution were run simultaneously (Hardstaff action in the U.S. (AOAC Official Method 991.26). The lower et al. 1990). detection level for the standard assay is ;0.5 ppm. This technique was used to validate the DA concentration in the HPLC Detection of Domoic Acid in Phytoplankton Using razor clams in this study. Briefly, 10–15 mL of the clarified Fluorenylmethoxycarbonyl (FMOC) Derivatization supernatant prepared as described above was transferred into a 25 mL disposable plastic syringe and filtered through 0.45 A more sensitive fluorescent fluorenylmethoxycarbonyl micron HA Millipore filter (Bedford, MA) into a labeled chloride (FMOC) derivatization method (Pocklington et al. scintillation vial. Salt clean-up was done with solid phase extrac- 1990) was used to determine particulate DA concentrations in tion columns (Hatfield et al. 1994). Strong anion exchange phytoplankton samples, which typically contained less DA than (SAX) solid phase extraction (SPE) cartridges (Whatman, shellfish tissues. The samples were processed on a Hewlett- Florham Park, NJ) were conditioned by washing successively Packard 1090 HPLC using a Vydac 201TP, 5 mm, 25 cm with 6 mL of methanol, 6 mL of deionized water, and 6 mL column, HP 1046A fluorescence detector, and column heater of 50% methanol. The SPE clean up also removes trypto- set to 40C with the following modification. In our analysis, phan, which is a major source of false positives in HPLC-UV solvents A (HPLC Water with 0.1% v/v TFA) and B (acetoni- detection of DA because it coelutes with DA. Each sample was trile with 0.1% v/v TFA) were pumped at 0.2 mL/min and the then drawn through a conditioned SAX SPE cartridge at a rate linear gradient elution was changed allowing for increased of 1 drop per second using a vacuum manifold. Flow was separation and resolution of the domoic acid peak. The initial stopped when the meniscus was just above the top of the gradient went from 70% A and 30% B at time of injection to

TABLE 1. Intertidal invertebrates sampled from several locations around Monterey Bay in November 2006. HPLC-UV analysis detected signiﬁcant levels of compounds comigrating with iso- and epi-domoic acid standards. These crude methanolic extracts were used to challenge the NOAA and Biosense ELISAs. The goal was to establish the extent to which the ELISA assays are confounded by the presence of coeluting compounds called as the domoic acid isomers epi- and iso-domoic acid by HPLC-UV assay. Nondetect samples are represented as 0 values.

Combined epi DA Concentration % Total DA DA Concentration % Total DA and iso-DA by NOAA Detected by by Biosennse Detected by Organism by HPLC (ppb) ELISA (ppb) NOAA ELISA ELISA (ppb) Biosense ELISA Chthamalus ﬁssus/dalli 281.7 0.00 0.00 0.02 0.01 Chthamalus ﬁssus/dalli 1,137.1 15.41 1.36 1.53 0.13 Littorina scutulata 198.7 10.57 5.32 3.02 1.52 Littorina scutulata 682.0 15.98 2.34 1.02 0.15 Littorina scutulata 119.5 0.00 0.00 0.17 0.14 Lottia digitalis 236.7 0.00 0.00 0.10 0.04 Lottia digitalis 477.9 13.91 2.91 0.09 0.02 Lottia digitalis 390.6 10.31 2.64 0.78 0.20 JOBNAME: jsr 27#5 2008 PAGE: 6 OUTPUT: Friday November 7 23:26:21 2008 tsp/jsr/176293/27-5-06

1306 LITAKER ET AL.

60% A and 40% B over 0–10 min, then held constant for 10 Data Analyses min; adjusted to 0% A and 100% B from 20–30 min, held Analytical results for DA concentrations determined from isocratic for 2 min; adjusted from 0% A and 100% B to 70% A razor clams, mussels, scallops and phytoplankton cells deter- and 30% B over 2 min, and then held constant at these (initial) mined by HPLC, FMOC-HPLC, LC-MS and the NOAA/MSI conditions until the end of the run at 45 min. Dihydrokainic ELISA were compared using linear regression analysis (Sokal & acid was used as an internal standard, as described by Rohlf 1995). The performance of the NOAA/MSI and Biosense Pocklington et al. (1990). ELISA kits was also compared using a subset of the phyto- A subset of phytoplankton samples was validated to confirm plankton samples. This comparison involved simultaneously the presence of DA (by mass) using liquid chromatography-mass analyzing phytoplankton extracts using the two kits and spectrometry (LC-MS) on a ThermoFinnigan Quantum Dis- comparing the results with those obtained using FMOC-HPLC. covery Max TSQ ESI Mass Spectrometer coupled to a HP 1100 All samples were run within a 24 h period to prevent differential series binary pump HPLC, following the general protocol of degradation of DA, which may occur in some samples. Data Quilliam et al. (1989a). Samples for LC-MS were prepared as for were compared using linear regression analysis. HPLC, but were then dried down under vacuum and redissolved in 100% methanol prior to injection. The HPLC conditions for the reverse phase were programmed for a linear gradient elution RESULTS AND DISCUSSION of 10:90% acetonitrile:deionized water (both containing 0.1% The NOAA/MSI ELISA accurately measured NRC stan- formic acid) up to 0:100% water:acetonitrile over 30 min. dard DA concentrations (Fig. 4) and gave equivalent results for razor clam (Fig. 5), mussel (Fig. 6), scallop (Fig. 7), and Testing Cross-Reactivity of the ELISA Against Glutamine, Kainic Acid phytoplankton extracts (Fig. 8) as obtained when using HPLC, and Putative Isomers Epi-DA and Iso-DA FMOC-HPLC, or LC-MS methods. When the variability in the Domoic acid is structurally similar to glutamine, glutamic NOAA/MSI ELISA and FMOC-HPLC method were com- acid and kainic acid, all of which can potentially co-occur with pared using replicate phytoplankton extracts they were found to DA in sample extracts (Fig. 1). To test for potential cross- be comparable (Fig. 9). The primary advantage of the NOAA/ reactivity with these compounds, the NOAA/MSI ELISA kit MSI ELISA over HPLC methods, besides a significantly lower was run using concentrations of glutamine, glutamic acid and cost per sample was much higher throughput. As many as 36 samples can be completed in <1.5 h after tissue extraction. kainic acid ranging from 10 ppb to 5 ppm. The ED50 for each The NOAA/MSI format was also flexible. An internal compound was calculated and then divided by ED50 for DA and multiplied by 100 to determine percent cross-reactivity (Table control was incorporated into each strip, which eliminated the 2). A majority of DA in razor clams and phytoplankton is in the necessity of running a standard curve each time the assay was form shown at the top of Figure 1. However, samples sometimes performed. Any unused strips could be removed and stored in a contain a larger quantity of compounds closely eluting with DA desiccator pouch at 4C for at least six months without on standard HPLC runs that have been identified as the DA compromising assay performance. This allowed as few as 3 conformers epi- and iso-DA (Wright et al. 1990, Kotaki et al. samples to be run in real time thereby avoiding the degradation 2005). To determine if the mAb used in this assay could detect of DA that can occur during storage, particularly once the these DA isomers, and the extent of interference by such samples have been extracted (Smith et al. 2006). For example, coeluting compounds present in crude extracts of intertidal when phytoplankton samples were run within 24 h using the barnacle, limpet, and snail samples, crude methanolic extracts Biosense ELISA kit, which has been validated by an interna- of these tissues were assays using HPLC-UV and both the tional collaborative study, and is officially approved by the NOAA/MSI and Biosense (Biosense Laboratories, Bergen, AOAC International for regulatory detection of DA in shellfish, Norway) ELISA methods. These intertidal invertebrate extracts exhibited high levels of the putative epi-DA and iso-DA isomers as called by comigration on HPLC chromatograms. These compounds are generally near detection limits in razor clams, crabs, and to a lesser extent in mussels, and therefore these extracts provided novel matrices for evaluating the accuracy of NOAA/MSI ELISA.

TABLE 2. Cross-reactivity of the NOAA/MSI ELISA with kainic acid. glutamine, and glutamic acid.

% Reactivity in the Domoic Analyte Acid Assay Domoic acid 100 Kainic acid 0.3 Figure 4. Relationship between various concentrations of National Glutamine <0.1 Research Council of Canada (NRC) domoic acid standards and the Glutamic acid <0.1 resultant NOAA/Mercury Science (NOAA/MSI) ELISA values determined using 10 different plates. JOBNAME: jsr 27#5 2008 PAGE: 7 OUTPUT: Friday November 7 23:26:22 2008 tsp/jsr/176293/27-5-06

DOMOIC ACID TEST KIT 1307

Figure 5. Domoic acid concentrations in razor clam tissues determined from replicate tissue extracts analyzed using HPLC and NOAA/Mercury Science (NOAA/MSI) ELISA. The inset shows an expanded version of the regression analysis for sample containing less than 2.5 ppm domoic acid. and the NOAA/MSI ELISA kit, equivalent results were small numbers of samples are being collected, they may have to obtained (Fig. 10, r2 ¼ 0.97). In contrast, when samples were be stored until a sufficient number of samples have been run two weeks apart the correlation dropped to r2 ¼ 0.79, accumulated to maximize the number of samples per kit. This indicating DA degradation. couldleadtosampledegradationandacriticaldelayin The ability to efficiently run a small number of samples in reporting when samples surpass the regulatory limit of 20 ppm. real time was not incorporated into other DA ELISA formats. Another advantage of the NOAA/MSI assay is that it could For example, the Biosense DA ELISA kit includes reagents for be run in either a quantitative or screening mode when assaying only two standard curves (product insert), therefore, only two shellfish tissues. For quantitative analysis, several dilutions were batches of samples can be run per kit. This means that when assayed simultaneously to obtain an accurate DA concentration.

Figure 6. Domoic acid concentrations in mussel tissues determined using Figure 7. Concentration of domoic acid in scallop tissues extracted from HPLC and the NOAA/Mercury Science (NOAA/MSI) ELISA. Aliquots the scallop (Pecten maximus) using the standard NOAA/Mercury Science from each sample were run simultaneously. (NOAA/MSI) protocol. JOBNAME: jsr 27#5 2008 PAGE: 8 OUTPUT: Friday November 7 23:26:27 2008 tsp/jsr/176293/27-5-06

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Figure 9. Comparison of the variability between phytoplankton extracts measured using FMOC-HPLC (h) and the NOAA/Mercury Science ELISA (¤). Error bars indicate %1 SD. Because the error bars largely overlap, for clarity, the standard deviation for the FMOC-HPLC is plotted in the horizontal direction and the NOAA ELISA in the vertical direction.

The NOAA/MSI and Biosense ELISA kits were tested against crude methanolic extracts of several intertidal invertebrates, which HPLC identiﬁed as containing >100 ppb levels of epi-DA and iso-DA. These compounds are reported to be less toxic DA congeners based on receptor binding assays (Sawant et al. 2007). Results from both ELISA kits revealed the presence of only trace amounts of DA equivalents in the extracts. The NOAA/MSI ELISA cross-reactivity with these compounds ranged from 0% to 5.3% and the Biosense ELISA cross- reactivity from 0.01% to 1.5% (Table 1) indicating that the ELISA assays are relatively insensitive to cogener interference. It should also be noted that the regulatory methods for assessing human safety are currently based on measuring DA Figure 8. (A) Domoic acid concentrations measured from split phyto- alone, not the combination of DA, iso-DA and epi-DA. These plankton sample extracts, which were measured within 24 h by FMOC- results indicated that both the NOAA/MSI and Biosense HPLC and either the NOAA/Mercury Science (NOAA/MSI) or Bio- sense ELISAs. (B) Comparison of domoic acid concentrations measured in split samples by either HPLC or ELISA.

Alternatively, to rapidly screen for DA concentrations of concern, the sample extracts were diluted 1:1,000 before running the assay. Taking into account the 1:10 dilution that occurred during the extraction process, the 1:1,000 dilution reduced samples in the 20 ppm DA range to ;2 ppb in the diluted sample. This concentration was within the linear range of the assay (0.1–3 ppb). Tissue samples with 5–10 fold less DA, and far below levels of concern, would show no detectible DA at this dilution. Tissues containing initial DA concentrations >30 ppm would be off scale and indicate a signiﬁcant DA concentration requiring action. Any samples from this rapid screening that were of concern could then be diluted and run again to obtain an accurate concentration. The NOAA/MSI ELISA test kit also comes with a simple Excel spreadsheet, which allowed the toxin concentrations to be quickly and easily calculated in either a quantitative or rapid screening mode. All that had to be Figure 10. NOAA/MSI ELISA versus Biosense ELISA for phytoplank- entered was the Bo (no DA added) and sample absorbance data ton samples when the two assays were run several weeks apart showing the from each strip, the weight of the extracted tissue samples, and increased variability, caused by differential degradation or absorption the extraction volumes. rates, when samples are not measured within the same 24 h period. JOBNAME: jsr 27#5 2008 PAGE: 9 OUTPUT: Friday November 7 23:26:32 2008 tsp/jsr/176293/27-5-06

DOMOIC ACID TEST KIT 1309

ELISA methods provide DA values comparable to the HPLC to qualify the 96 well plate format for regulatory use by public values currently used as a basis for regulatory decisions. health officials. We are also developing a field test kit that can be Measuring low concentrations of DA in real time is partic- used to detect DA levels in shellfish tissues above or below 20 ularly important because the presence or absence of DA ppm within 10 min after extraction. The test will require no contamination is frequently patchy and associated with variable laboratory equipment other than a homogenizer and can be onshore transport of toxic phytoplankton blooms (e.g., Trainer used directly in the field by non-technical personnel, including et al. 2002). Depending on prevailing winds and currents, one shellfish harvesters and members of citizen monitoring groups harvest area can become highly contaminated over a short period and local volunteers. whereas adjacent regions remain uncontaminated (Trainer et al. In summary, the NOAA/MSI ELISA test kit provides an 2000). These differentially affected regions frequently include accurate, flexible and cost effective method for measuring DA areas where significant commercial and recreational clam har- in clam, mussel and scallop tissues, as well as in phytoplankton vests occur. This variability complicates monitoring programs samples. The assay yields concentrations for DA that are designed to protect human health. The current standard practice indistinguishable from those obtained by HPLC. With further involves shipping shellfish samples to a centralized facility for validation, the NOAA/MSI ELISA kit is expected to be HPLC analyses, introducing delays between 3–14 days from the approved as a regulatory method for making decisions con- date of sample collection to reporting results. This turnaround cerning public health. The short assay (1.5-h) processing time, time is too slow to adequately protect subsistence shellfish and relatively low cost, compared with HPLC analysis, mean harvesters who rely on clams consumed within a day or two of that the ELISA can be used in more remote locations by harvest. The cost of HPLC analysis is also relatively high per environmental managers and public health officials to provide sample and requires a substantially higher capital investment near real-time monitoring capacities. compared with the NOAA/MSI ELISA method. Having an economical technique for better assessing the degree of contam- ACKNOWLEDGMENTS ination locally, and in real time, is of great value for local resource managers and public health officials. The authors thank Mitch Lesoing and Mel Moon of the The ability to detect DA in phytoplankton using the NOAA/ Quileute Tribe whose initial request for development of a MSI kits would further benefit environmental monitoring domoic test kit and constant encouragement were key to the programs designed to detect the early onset of toxic Pseudo- success of the test kit. This work was supported by a competitive nitzschia blooms. It is known that increases in the Pseudo-nitzschia grant awarded by the MERHAB program specifically for assay capable of producing DA often precedes the contamination of development as well as a NOAA NCCOS Ecology and Ocean- shellfish and other filter feeders by a week or two (Trainer & ography of Harmful Algal Blooms (ECOHAB) grant GAD# Suddleson 2005). A combination of cell counts and direct R83-1705) and two Monitoring and Event Response for Harm- toxicity measurements should provide timely predictions for ful Algal Blooms (MERHAB) grants: NA04NOS4780239-03 marine resource managers and public health officials. The kit is and NA05NOS4781228. J. Bastion and A. Odell’s participation now commercially available with MSI authorized to market, was funded by a surcharge to the Washington State shellfish manufacture and distribute the 96-well plate format test kits. license provided or ORHAB. Jonathan Deeds provided helpful We anticipate completing the necessary validation procedures edits and suggestions.

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